Correlation of LMP10 Expression and Clinical Outcome in Human Papillomavirus (HPV) Positive and HPV-Negative Tonsillar and Base of Tongue Cancer

Aim

To examine LMP10 expression and its possible impact on clinical outcome in human papillomavirus (HPV) positive and HPV-negative tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC).

Background

Outcome is better in HPV-positive TSCC and BOTSCC compared to matching HPV-negative tumours, with roughly 80% vs. 40% 5-year disease free survival (DFS) with less aggressive treatment than today’s chemoradiotherapy. Since current treatment often results in harmful side effects, less intensive therapy, with sustained patient survival would be an attractive alternative. However, other markers together with HPV status are necessary to select patients and for this purpose LMP10 expression is investigated here in parallel to HPV status and clinical outcome.

Materials and Methods

From 385 patients diagnosed between 2000 and 2007 at the Karolinska University Hospital, 278 formalin fixed paraffin embedded TSCC and BOTSCC biopsies, with known HPV DNA status, were tested for LMP10 nuclear and cytoplasmic expression (fraction of positive cells and staining intensity). The data was then correlated to clinical outcome.

Results

An absent/low compared to a moderate/high LMP10 nuclear fraction of positive cells was correlated to a better 3-year DFS in the HPV-positive group of patients (log-rank p = 0.005), but not in the HPV-negative group. In the HPV-negative group of patients, in contrast to the HPV-positive group, moderate/high LMP10 cytoplasmic fraction and weak/moderate/high LMP10 cytoplasmic intensity correlated to a better 3-year DFS (p = 0.003 and p = 0.001) and 3-year overall survival (p = 0.001 and 0.009).

Conclusion

LMP10 nuclear expression in the HPV-positive group and LMP10 cytoplasmic expression in the HPV-negative group of patients correlated to better clinical outcome.     

Human Papillomavirus in Head and Neck Squamous Cell Carcinoma of Unknown Primary Is a Common Event and a Strong Predictor of Survival

Background

The purpose of this study was to examine the prevalence of human papillomavirus (HPV) in patients with head and neck squamous cell carcinoma of unknown primary (CUP).

Methods

All patients diagnosed with and treated for CUP between January 1, 2000, and June 1, 2011, at two Danish medical centers were included. All patients received a thorough diagnostic work-up, including FDG-PET, before being diagnosed as CUP. We determined the HPV status in all patients using a combination of HPV DNA PCR and p16 stain. In addition, clinical information on the study patients was retrieved from clinical records.

Results

Of the identified 60 patients with CUP, 13 were shown to be positive for HPV DNA, amounting to 22% of the study population. In addition, we were able to show a clear disease-free and overall-survival benefit in the HPV-positive group, with a hazard ratio of 0.16 (95% CI: 0.038–0.67) for over-all survival. This survival benefit was also apparent when adjusted for advanced age in a multivariate Cox regression analysis.

Conclusion

A fairly large percentage of CUP cases are HPV-related, and because this is related to both the location and prognosis, we recommend HPV testing as part of the diagnostic work-up.     

HPV+/-宫颈癌患者临床特点与预后的对比分析

目的:探讨HPV阴性与HPV阳性宫颈癌的临床特点及其和预后的关系。方法:选取住院手术的HPV呈阴性与阳性的宫颈癌患者各86例,且两组患者在年龄、FIGO临床分期相近。分析两组患者的年龄、肿瘤临床分期、病理分型、原发肿瘤大小、分化程度、基层浸润深度、淋巴结转移、治疗方案情况及其5年RFS和总生存时间。结果:两组间宫颈癌患者肿瘤分化程度、肌层浸润深度、淋巴结转移间差异有统计学意义(P0.05)。HPV阳性组和HPV阴性组随访时间分别为63(13~87)个月、61(9~90)个月,5年总生存率分别为70.4%和61.3%,平均总生存时间分别为(73.15±2.74)月和(62.72±3.03)月,差异有统计学意义(p0.05)。HPV阴性组和HPV阳性组5年RFS分别为52.3%和66.4%,平均RFS为(51.57±4.62)月和(58.83±3.46)月,差异有统计学意义(p0.05)。结论:HPV阴性宫颈癌瘤体倾向于低分化,易发生局部侵犯和淋巴结转移,其临床预后较差。     

Human Papillomavirus Prevalence in Invasive Laryngeal Cancer in the United States

Purpose

Human papillomavirus (HPV) is a major risk factor for specific cancers of the head and neck, particularly malignancies of the tonsil and base of the tongue. However, the role of HPV in the development of laryngeal cancer has not been definitively established. We conducted a population-based, cancer registry study to evaluate and characterize the genotype-specific prevalence of HPV in invasive laryngeal cancer cases diagnosed in the U.S.

Methods

The presence of genotype-specific HPV DNA was evaluated using the Linear Array HPV Genotyping Test and the INNO-LiPA HPV Genotyping Assay in formalin-fixed paraffin embedded tissue from 148 invasive laryngeal cancer cases diagnosed in 1993–2004 within the catchment area of three U.S. SEER cancer registries.

Results

HPV DNA was detected in 31 of 148 (21%) invasive laryngeal cancers. Thirteen different genotypes were detected. Overall, HPV 16 and HPV 33 were the most commonly detected types. HPV was detected in 33% (9/27) of women compared with 18% (22/121) of men (p = 0.08). After adjustment for age and year of diagnosis, female patients were more likely to have HPV-positive laryngeal tumors compared to males (adjusted OR 2.84, 95% CI 1.07–7.51). Viral genotype differences were also observed between the sexes. While HPV 16 and 18 constituted half of HPV-positive cases occurring in men, among women, only 1 was HPV 16 positive and none were positive for HPV 18. Overall 5-year survival did not vary by HPV status.

Conclusions

HPV may be involved in the development of a subset of laryngeal cancers and its role may be more predominant in women compared to men.     

Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells

ObjectiveTo map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients.ResultsHypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5′-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).ConclusionsOur study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.     

Human Papillomavirus Infection in 674 Chinese Patients with Laryngeal Squamous Cell Carcinoma

Objectives

Previous reports suggest a strong association between human papillomavirus (HPV) and the etiology of laryngeal squamous cell carcinoma (LSCC). However, clinical data regarding the HPV infection rate among LSCC patients remain largely inconsistent.

Methods

In total, 674 LSCC patients from three major hospitals in Shanghai were enrolled in this study. We determined the patients'' HPV infection status using immunohistochemistry and the GenoArray HPV genotyping assay and calculated their long-term survival rate using the Kaplan-Meier method.

Results

The total P16-positive rate according to immunostaining results was 7.57% (51/674). None of the P16-negative patients were HPV-positive according to the HPV genotyping test. The rate of HPV infection among patients with LSCC was 4.9% (33/674). HPV infection was more common among nonsmokers (P<0.05), nondrinkers (P<0.05), and patients with supraglottic LSCC (P<0.05). Of the 33 HPV-positive patients, 28 (84.8%) were infected with HPV-16, 2 with HPV-18, 1 with HPV-31, 1 with HPV-33 and 1 with HPV-45. The 3-year overall survival rate and progression-free survival rate were higher in HPV-positive than HPV-negative patients, but the difference was not statistically significant (76.3% vs. 70.7%, P = 0.30 and 65.1% vs. 58.3%, P = 0.37, respectively).

Conclusion

HPV was not a main causal factor in LSCC carcinogenesis in this Chinese population. HPV infection did not alter patients'' overall survival or progression-free survival rates in this study.     

The Prognostic Value of HPV Status and p16 Expression in Patients with Carcinoma of the Anal Canal

Background

In anal cancer studies, the detection frequency of high-risk HPV (human papillomavirus) is variable, depending on the method used. There are limited data reporting results of different HPV detection techniques in the same clinical series, and very few correlating results with clinical outcome.

Objectives

To evaluate tumor expression of p16/HPV16 using three different methods, and to determine their association with clinical outcome in patients with anal canal squamous cell carcinomas (SCC).

Design

This retrospective study included patients with anal canal SCC treated with definitive radiotherapy or chemoradiotherapy at a single institution between 1992 and 2005. Formalin-fixed paraffin–embedded tumor samples from 53 of the 89 (60%) patient pre-treatment biopsies were adequate for tissue microarray construction. HPV status was determined using: p16 expression by conventional immunohistochemistry (IHC) and quantitative IHC (AQUA), HPV genotype analysis by chromogenic in situ hybridization (CISH) and HPV linear array sub-typing. Expression status was correlated with clinical outcome.

Results

80% (28/35) of patient tumors had high p16 expression using conventional IHC. HPV16 CISH was positive in 81% (34/42) of tumors, and 78% (28/36) of tumors were HPV subtype 16. HPV16 CISH correlated with p16 evaluated by conventional IHC (correlation coefficient 0.46; p = 0.01) and by p16 AQUA score (correlation coefficient 0.49; p = 0.001). A subset of cases (15%) had very high p16 quantitative IHC scores (>244) and were associated with a higher incidence of local or distant recurrence (p = 0.04).

Conclusions

The vast majority (80%) of anal canal SCC in our series were positive for HPV16/p16, regardless of the testing method used. The exploratory analysis of automated quantitative IHC scoring was the only technique to define a subset of patients with a worse prognosis by p16 expression status on univariate analysis. Further exploration of the molecular mechanisms of treatment resistance in association with very high p16 expression is warranted.     

Pathobiological Implications of Mucin (MUC) Expression in the Outcome of Small Bowel Cancer

Mucins have been associated with survival in various cancer patients, but there have been no studies of mucins in small bowel carcinoma (SBC). In this study, we investigated the relationships between mucin expression and clinicopathologic factors in 60 SBC cases, in which expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6 and MUC16 in cancer and normal tissues were examined by immunohistochemistry. MUC1, MUC5AC and MUC16 expression was increased in SBC lesions compared to the normal epithelium, and expression of these mucins was related to clinicopathologic factors, as follows: MUC1 [tumor location (p = 0.019), depth (p = 0.017) and curability (p = 0.007)], MUC5AC [tumor location (p = 0.063) and lymph node metastasis (p = 0.059)], and MUC16 [venous invasion (p = 0.016) and curability (p = 0.016)]. Analysis of 58 cases with survival data revealed five factors associated with a poor prognosis: poorly-differentiated or neuroendocrine histological type (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p = 0.026), venous invasion (p<0.001) and curative resection (p<0.001), in addition to expression of MUC1 (p = 0.042), MUC5AC (p = 0.007) and MUC16 (p<0.001). In subsequent multivariate analysis with curability as the covariate, lymph node metastasis, venous invasion, and MUC5AC and/or MUC16 expression were significantly related to the prognosis. Multivariate analysis in curative cases (n = 45) showed that SBC with MUC5AC and/or MUC16 expression had a significantly independent high hazard risk after adjusting for the effects of venous invasion (hazard ratio: 5.6, 95% confidence interval: 1.8–17). In conclusion, the study shows that a MUC5AC-positive and/or MUC16-positive status is useful as a predictor of a poor outcome in patients with SBC.     

Characteristics and Prognostic Implications of High-Risk HPV-Associated Hypopharyngeal Cancers

Background

High-risk human papillomavirus (HPV) is an oncogenic virus that causes oropharyngeal cancers, and it has a favorable outcome after the treatment. Unlike in oropharyngeal cancer, the prevalence and role of high-risk HPV in the etiology of hypopharyngeal squamous cell carcinoma (HPSCC) is uncertain.

Objective

The aim of the present study was to evaluate the effect and prognostic significance of high-risk HPV in patients with HPSCC.

Methods

The study included 64 subjects with HPSCC who underwent radical surgery with or without radiation-based adjuvant therapy. Primary tumor sites were the pyriform sinus in 42 patients, posterior pharyngeal wall in 19 patients, and postcricoid area in 3 patients. High-risk HPV in situ hybridization was performed to detect HPV infection.

Results

The positive rate of high-risk HPV in situ hybridization was 10.9% (7/64). There was a significant difference in the fraction of positive high-risk HPV among pyriform sinus cancer (16.7%), posterior pharyngeal wall cancer (0%), and postcricoid area cancer (0%) (p = 0.042). The laryngoscopic examination revealed a granulomatous and exophytic appearance in 85.7% (6/7) of patients with high-risk HPV-positive pyriform sinus cancer, but in only 31.4% (11/35) of patients with high-risk HPV-negative pyriform sinus cancer (p = 0.012). Significant correlations were found between positive high-risk HPV and younger age (p = 0.050) and non-smoking status (p = 0.017). HPV-positive patients had a significantly better disease-free survival (p = 0.026) and disease-specific survival (p = 0.047) than HPV-negative patients.

Conclusions

High-risk HPV infection is significantly related to pyriform sinus cancer in patients with HPSCC.     

Prognostic Significance of Overexpressed p16INK4a in Patients with Cervical Cancer: A Meta-Analysis

Background

p16^INK4a is a tumor suppressor protein which is induced in cells upon the interaction of high-risk HPV E7 with the retinoblastoma protein by a positive feedback loop, but cannot exert its suppressing effect. Previous reports suggested that p16^INK4a immunostaining allows precise identification of even small CIN or cervical cancer lesions in biopsies. The prognostic value of overexpressed p16^INK4a in cervical cancer has been evaluated for several years while the results remain controversial. We performed a systematic review and meta-analysis of studies assessing the clinical and prognostic significance of overexpression of p16^INK4a in cervical cancer.

Methods

Identification and review of publications assessing clinical or prognostic significance of p16^INK4a overexpression in cervical cancer until March 1, 2014. A meta-analysis was performed to clarify the association between p16^INK4a overexpression and clinical outcomes.

Results

A total of 15 publications met the criteria and comprised 1633 cases. Analysis of these data showed that p16^INK4a overexpression was not significantly associated with tumor TNM staging (I+II vs. III+IV) (OR = 0.75, 95% confidence interval [CI]: 0.35–1.63, P = 0.47), the tumor grade (G1+ G2 vs. G3) (OR = 0.78, 95% CI: 0.39–1.57, P = 0.49), the tumor size (<4 vs. ≥4 cm) (OR = 1.10, 95% CI: 0.45–2.69, P = 0.83), or vascular invasion (OR = 1.20, 95% CI: 0.69–2.08, P = 0.52). However, in the identified studies, overexpression of p16^INK4a was highly correlated with no lymph node metastasis (OR = 0.51, 95% CI: 0.28–0.95, P = 0.04), increased overall survival (relative risk [RR]: 0.42, 95% CI: 0.24–0.72, P = 0.002) and increased disease free survival (RR: 0.60, 95% CI: 0.44–0.82, P = 0.001).

Conclusions

This meta-analysis shows overexpression of p16^INK4a in cervical cancer is connected with increased overall and disease free survival and thus marks a better prognosis.     

Differences in Oral Sexual Behaviors by Gender,Age, and Race Explain Observed Differences in Prevalence of Oral Human Papillomavirus Infection

Purpose

This study explores whether gender, age and race differences in oral sexual behavior account for the demographic distribution of oral human papillomavirus infection (HPV) and HPV-positive oropharyngeal cancer (HPV-OSCC)

Methods

This analysis included 2,116 men and 2,140 women from NHANES (2009–10) who answered a behavioral questionnaire and provided an oral-rinse sample for HPV detection. Weighted prevalence estimates and prevalence ratios (PR) were calculated for sexual behaviors and oral HPV infection by gender, age-cohort (20–29, 30–44, 45–59, 60–69), and race, and contrasted with incidence rate ratios (IRR) of OSCC from SEER 2009. Multivariate logistic regression was used to evaluate predictors of oral sexual behavior and oral HPV16 infection.

Results

Differences in oral sexual behavior were observed by gender, age-cohort and race. Most men (85.4%) and women (83.2%) had ever performed oral sex, but men had more lifetime oral and vaginal sexual partners and higher oral HPV16 prevalence than women (each p<0.001). 60–69 year olds (yo) were less likely than 45–59 or 30–44 (yo) to have performed oral sex (72.7%, 84.8%, and 90.3%, p<0.001), although oral HPV16 prevalence was similar. Prevalence ratios (PR) of ever oral sex in men vs. women (PR = 1.03), and 45–59 vs. 30–44 year-old men (PR = 0.96) were modest relative to ratios for oral HPV16 infection (PRs = 1.3–6.8) and OSCC (IRR = 4.7–8.1). In multivariate analysis, gender, age-cohort, and race were significant predictors of oral sexual behavior. Oral sexual behavior was the primary predictor of oral HPV16 infection; once this behavior was adjusted for, age-cohort and race were no longer associated with oral HPV16.

Conclusion

There are differences in oral sexual behaviors when considering gender, age-cohort and race which explain observed epidemiologic differences in oral HPV16 infection across these groups.     

Clinical Relevance of Loss of 11p15 in Primary and Metastatic Breast Cancer: Association with Loss of PRKCDBP Expression in Brain Metastases

The occurrence of brain metastases among breast cancer patients is currently rising with approximately 20–25% incidence rates, underlining the importance of the identification of new therapeutic and prognostic markers. We have previously screened for new markers for brain metastasis by array CGH. We found that loss of 11p15 is common among these patients. In this study, we investigated the clinical significance of loss of 11p15 in primary breast cancer (BC) and breast cancer brain metastases (BCBM). 11p15 aberration patterns were assessed by allelic imbalance (AI) analysis in primary BC (n = 78), BCBM (n = 21) and metastases from other distant sites (n = 6) using six different markers. AI at 11p15 was significantly associated with BCBM (p = 0.002). Interestingly, a subgroup of primary BC with a later relapse to the brain had almost equally high AI rates as the BCBM cases. In primary BC, AI was statistically significantly associated with high grade, negative hormone receptor status, and triple-negative (TNBC) tumors. Gene expression profiling identified PRKCDBP in the 11p15 region to be significantly downregulated in both BCBM and primary BC with brain relapse compared to primary tumors without relapse or bone metastasis (fdr<0.05). qRT-PCR confirmed these results and methylation was shown to be a common way to silence this gene. In conclusion, we found loss at 11p15 to be a marker for TNBC primary tumors and BCBM and PRKCDBP to be a potential target gene in this locus.     

Significance of HPV-58 Infection in Women Who Are HPV-Positive,Cytology-Negative and Living in a Country with a High Prevalence of HPV-58 Infection

Purpose

Cervical cytology and human papillomavirus (HPV) DNA co-testing is recommended as a screening method for detecting cervical lesions. However, for women who are HPV-positive but cytology-negative, the appropriate management and significance of HPV-58 infection remain unknown.

Methods

This study of prevalent HPV detected at baseline with a median follow-up of 3.2 years evaluated the risk factors associated with cervical abnormalities and assessed the significance of HPV-58 infection. A total of 265 women were enrolled. All high-grade squamous intraepithelial lesions (HSIL) that were detected by cytology were confirmed by histology. Histological diagnoses of cervical intraepithelial neoplasia 2/3 were classified as HSIL. Women were classified into four groups according to the HPV genotype that was detected at their first visit: HPV-58 (n = 27), HPV-16 (n = 52; 3 women had HPV-58 co-infection), ten other high risk (HR) types (n = 79), or low/undetermined risk types (n = 107).

Results

Of 265 women, 20 (7.5%) had HSIL on their follow-up examinations. There were significant differences in the cumulative incidence of HSIL between the four groups (p<0.001). The 5-year cumulative incidence rates of HSIL were 34.0% (95% CI: 17.3–59.8%) in HPV-58 positive cases, 28.0% (95% CI: 13.8–51.6) in HPV-16 positive cases, 5.5% (95% CI: 2.1–14.0%) in one of the ten other types of HR-HPV positive cases, and 0% in women with low/undetermined risk HPV. When seen in women with HR-HPV (n = 158), persistent HPV infection was a significant factor associated with the development of HSIL (hazard ratio = 15.459, 95% CI: 2.042–117.045). Women with HPV-58 had a higher risk (hazard ratio = 5.260, 95% CI: 1.538–17.987) for the development of HSIL than women with HPV-16 (hazard ratio = 3.822, 95% CI: 1.176–12.424) in comparison with women with other types of HR-HPV.

Conclusion

HPV-58 has a high association with the development of HSIL in women who are HPV-positive and cytology-negative.     

Impact of human papilloma virus infection on the response of head and neck cancers to anti-epidermal growth factor receptor antibody therapy

Infection with human papillomaviruses (HPVs) characterizes a distinct subset of head and neck squamous cell cancers (HNSCCs). HPV-positive HNSCC preferentially affect the oropharynx and tonsils. Localized HPV-positive HNSCCs have a favorable prognosis and treatment outcome. However, the impact of HPV in advanced or metastatic HNSCC remains to be defined. In particular, it is unclear whether HPV modulates the response to cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), which is a mainstay of treatment of advanced HNSCC. To this end, we have examined the sensitivity of HPV-positive and -negative HNSCC models to cetuximab and cytotoxic drugs in vitro and in vivo. In addition, we have stably expressed the HPV oncogenes E6 and E7 in cetuximab-sensitive cancer cell lines to specifically investigate their role in the antibody response. The endogenous HPV status or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. Cetuximab effectively inhibited the growth of E6- and E7-expressing tumors grafted in NOD/SCID mice. In support, formalin-fixed, paraffin-embedded tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16^INK4a expression, an established biomarker of HPV infection. Response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16^INK4A-positive and p16^INK4A-negative tumors. In conclusion, HPV oncogenes do not modulate the anti-EGFR antibody response in HSNCC. Cetuximab treatment should be administered independently of HPV status.     

Loss of the α2β1 integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro

Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.     

Estimating the conditional probability of developing human papilloma virus related oropharyngeal cancer by combining machine learning and inverse Bayesian modelling

The epidemic increase in the incidence of Human Papilloma Virus (HPV) related Oropharyngeal Squamous Cell Carcinomas (OPSCCs) in several countries worldwide represents a significant public health concern. Although gender neutral HPV vaccination programmes are expected to cause a reduction in the incidence rates of OPSCCs, these effects will not be evident in the foreseeable future. Secondary prevention strategies are currently not feasible due to an incomplete understanding of the natural history of oral HPV infections in OPSCCs. The key parameters that govern natural history models remain largely ill-defined for HPV related OPSCCs and cannot be easily inferred from experimental data. Mathematical models have been used to estimate some of these ill-defined parameters in cervical cancer, another HPV related cancer leading to successful implementation of cancer prevention strategies. We outline a “double-Bayesian” mathematical modelling approach, whereby, a Bayesian machine learning model first estimates the probability of an individual having an oral HPV infection, given OPSCC and other covariate information. The model is then inverted using Bayes’ theorem to reverse the probability relationship. We use data from the Surveillance, Epidemiology, and End Results (SEER) cancer registry, SEER Head and Neck with HPV Database and the National Health and Nutrition Examination Surveys (NHANES), representing the adult population in the United States to derive our model. The model contains 8,106 OPSCC patients of which 73.0% had an oral HPV infection. When stratified by age, sex, marital status and race/ethnicity, the model estimated a higher conditional probability for developing OPSCCs given an oral HPV infection in non-Hispanic White males and females compared to other races/ethnicities. The proposed Bayesian model represents a proof-of-concept of a natural history model of HPV driven OPSCCs and outlines a strategy for estimating the conditional probability of an individual’s risk of developing OPSCC following an oral HPV infection.     

Reduced Expression of the Antigen Processing Machinery Components TAP2, LMP2, and LMP7 in Tonsillar and Base of Tongue Cancer and Implications for Clinical Outcome

OBJECTIVES: Patients with human papillomavirus (HPV)–positive tonsillar squamous cell carcinoma (TSCC) and base of tongue squamous cell carcinoma (BOTSCC) have a better clinical outcome than those with corresponding HPV-negative tumors. Moreover, there is a strong positive correlation between absent/low as opposed to strong HLA class I expression and favorable clinical outcome for HPV-positive tumors, while the reverse applies to HPV-negative tumors. The expression of the antigen processing machinery (APM) components TAP1, TAP2, LMP2, and LMP7 in these tumors in relation to HPV status, HLA class I expression, each other, and clinical outcome was therefore investigated. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded TSCC and BOTSCC, derived from 151 patients and previously analyzed for HPV DNA, HLA class I, and LMP10 expression were stained by immunohistochemistry for TAP1, TAP2, LMP2, and LMP7. RESULTS: Absent/low TAP2, LMP2, and LMP7 expression, similar to HLA class I and LMP10, was common in TSCC and BOTSCC, irrespective of HPV status. Expression of TAP1 and TAP2 was correlated, as was LMP2 to LMP7. LMP2 and LMP7 expression was also associated to HLA class I expression. Moreover, absence of LMP7 was linked to increased disease-free survival in both HPV-positive and HPV-negative cases. CONCLUSION: Reduced expression of TAP2, LMP2, and LMP7 was frequent in TSCC and BOTSCC and their expression as well as that of TAP1 was often interrelated. Furthermore, low LMP7 expression correlated to better clinical outcome and may, together with HPV status, potentially be used for prediction of treatment response.     

Correlation of STAT1 with Apoptosis and Cell-Cycle Markers in Esophageal Squamous Cell Carcinoma

We recently found evidence that STAT1 in esophageal squamous carcinoma (ESCC) cells exerts tumor suppressor function, and it regulates five key regulators of apoptosis or cell-cycle progression, including Bcl-2, Bcl-xL, survivin, cyclin D1 and p21. In this study, we confirmed these findings in four ESCC cell lines. Using immunohistochemistry, we also assessed the expression of these proteins in 62 primary tumors. The expression of these markers was heterogeneous, ranging 39 to 69% of the cohort. Significant correlation was found between STAT1 and three proteins (p21, Bcl-xL and survivin), whereas only a trend was identified for cyclin D1 and Bcl-2. We then correlated the expression of these proteins with several clinicopathologic parameters including lymph node metastasis, depth of invasion, clinical stage and overall survival. Significant correlations were found between Bcl-2 and deep invasion (p = 0.033), survivin and lymph node metastasis (p = 0.006), as well as cyclin D1 and clinical stage (p = 0.014). Patients with p21-positive tumors had a significantly longer survival compared to those with p21-negative tumors (p = 0.031). To conclude, our findings support the concept that STAT1 exerts its tumor suppressor effects in ESCC via modulating the expression of key regulators of apoptosis and cell-cycle progression.     

Alcohol Consumption and Viral Load Are Synergistically Associated with CIN1

Purpose

We investigated the association between alcohol consumption and risk of cervical intraepithelial neoplasia (CIN) and cervical cancer, and determined whether these associations were modified by human papillomavirus (HPV) viral load in high-risk HPV-positive women participating in the Korean HPV cohort study (KHPV).

Methods

Among the women recruited in the KHPV (n = 1,243) from March 2006 to December 2009, we analyzed normal cytology (n = 581) as control group, CIN1 (n = 299), CIN2/3 (n = 161), or cervical cancer (n = 202). Multinomial logistic analysis was performed to estimate multivariate-adjusted odds ratios (OR).

Results

Alcohol drinkers had an increased risk of CIN1 (OR = 2.18, 95% CI 1.22–3.89) compared with non-drinkers after adjusting for potential confounders. Subjects with more frequent alcohol consumption had a higher risk of CIN1 (p for linear trend <0.0001). Higher ethanol consumption was associated with an increased risk of CIN1 (p for linear trend = 0.0001). We also observed a synergistic effect between HPV viral load and alcohol consumption: drinkers with a high HPV viral load (≥100 RLU/PC) were associated with a significantly increased risk of CIN1 (OR = 19.1; 95% CI, 6.60–55.3, interaction p<0.001). There were no associations between alcohol drinking and CIN2/3 or cervical cancer.

Conclusions

HPV viral load and alcohol was associated with the risk of CIN1 among high-risk HPV-positive women. This is the first demonstration that alcohol is an independent and combined risk factor of CIN1.     

Differential Expression of HPV16 L2 Gene in Cervical Cancers Harboring Episomal HPV16 Genomes: Influence of Synonymous and Non-Coding Region Variations

We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4–6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1–2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.